Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components

ABSTRACT

The invention encompasses systems for column-based separations, methods of packing and unpacking columns and methods of separating components of samples. In one aspect, the invention includes a method of packing and unpacking a column chamber, comprising: a) packing a matrix material within a column chamber to form a packed column; and b) after the packing, unpacking the matrix material from the column chamber without moving the column chamber. In another aspect, the invention includes a system for column-based separations, comprising: a) a fluid passageway, the fluid passageway comprising a column chamber and a flow path in fluid communication with the column chamber, the flow path being obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid, the flow path extending through the column chamber and through the retaining material, the flow path being configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path; and b) the fluid passageway extending through a valve intermediate the column chamber and the retaining material.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of application Ser.No. 09/087,454 filed May 27, 1998.

TECHNICAL FIELD

[0002] The invention pertains to systems for column-based separation,and to methods of forming and utilizing packed columns. In specificembodiments, the invention pertains to methods of separating samplecomponents.

BACKGROUND OF THE INVENTION

[0003] Column-based separations are frequently used for selectivelyremoving components from mixtures. A first step in utilizingcolumn-based technology is to form a column. Such can be accomplishedwithin a column chamber. An exemplary prior art column chamber 10 isillustrated in FIG. 1. Column chamber 10′ comprises a longitudinaltubular section 12 having ends 14 and 16. An inlet 18 is provided at end16, and an outlet 20 is provided at end 14. Outlet 20 is obstructed by aporous filter 22. Filter 22 can comprise, for example, a porous frittedglass or ceramic material.

[0004] A packed column is formed within chamber 10 by flowing a slurrycomprising a mixture of matrix material 15 and carrier fluid 17 intoinlet 18. Matrix material 15 comprises a plurality of particulates, suchas, for example, beads. Filter 22 is permeable to the carrier fluid andimpermeable to the matrix material. Accordingly, as the slurry is flowedinto column chamber 10, matrix material 15 stacks against filter 22 toform a packed column 19 within tubular portion 12.

[0005] The composition of carrier fluid 17 and matrix material 15 varydepending on the components that are intended to be separated by thepacked column, and on the mixtures (samples) within which suchcomponents are found. Example matrix materials are Sr-resin, TRU-resin,and TEVA-resin, all of which can be obtained from EIChrom Industries,Inc., of Darien, Ill. Such matrix materials can have particle sizes inthe range of, for example, 20-100 micrometers. Sr-resin, TRU-resin, andTEVA-resin can be used for, for example, selectively retainingradioactive materials. Specifically, Sr-resin can selectively retainstrontium, TRU-resin can selectively retain americium, and TEVA-resincan selectively retain technetium. Slurries utilized for forming packedcolumns of Sr-resin, TEVA-resin, or TRU-resin can comprise, for example,0.074 gram/mL of Sr-resin in 3 M HNO₃; 0.142 grams/mL of TEVA-resin in 4M HNO₃; or 0.076 grams/mL of TRU-resin in 0.1 M HNO₃, respectively.

[0006] Other materials that can be separated utilizing column-basedsystems are, for example, biological materials, such as nucleic acids.For instance, Tepnel Life Sciences sells polymeric micro-beads indiameters of approximately 60-100 micrometers which are covalentlylinked to specific oligonucleotide capture probes. Such micro-beads canbe utilized for selective purification of nucleic acid fragments from abiological sample. For purposes of interpreting this disclosure and theclaims that follow, the term “nucleic acid” is defined to include DNAnucleotides and RNA nucleotides, as well as any length polymercomprising DNA nucleotides or RNA nucleotides.

[0007] In addition to the above-discussed exemplary uses forcolumn-based separations, numerous other applications for column-basedseparations are known to persons of ordinary skill in the art. Thecolumn-based separations generally have in common that a mixture in afirst physical state (typically either a gas phase or a liquid phase) isflowed across a column matrix in a second physical state (typicallyeither a liquid phase or a solid phase) to separate a component of themixture from other materials of the mixture. Accordingly, the physicalstate of the matrix is generally different than the physical state ofthe component that is to be separated.

[0008] It can be desired to quantitate and/or otherwise analyze anamount of a component retained by a column matrix in a packed column.Accordingly, it can be desired to extract a retained component from amatrix materia. A method of extracting a retained component is tosubject the column matrix to conditions which disrupt interactionsbetween the matrix material and the component to thereby elute thecomponent from the matrix material. In some applications, it isdesirable to elute the retained material from the matrix material whilethe matrix material is still within a packed column, and in otherapplications it is desirable to remove the matrix material from a packedcolumn before eluting the retained component. Additionally, there aresome applications in which it is desirable to remove a matrix materialfrom a packed column and thereafter analyze the matrix material directlyto quantitate and/or otherwise analyze an amount of a component retainedon the matrix material.

[0009] A difficulty in utilizing column-based separations is in removingmatrix material from a column chamber and subsequently repackingadditional matrix material in the chamber to re-form a packed column.There are numerous reasons for removing matrix material from a chamber.For instance, a matrix material of a packed column can be renderedunusable after an initial separation, or after an initial series ofseparations. A matrix material can be rendered unusable if it isdegraded by fluids passed through the material during a separation.Also, the matrix material can be rendered unusable if it becomescontaminated by materials within a sample because such contamination canpose a risk of cross-contamination.

[0010] For one or more of the above-discussed reasons, it is frequentlydesirable to repeatedly pack and unpack a column chamber with matrixmaterial. Because packing and unpacking of column chambers is atime-consuming and laborious process, disposable columns are generallyused. However, disposable columns still require labor for columnchangeout. Accordingly, it is desirable to develop new methods forpacking and unpacking column chambers.

[0011] A recent improvement is described with reference to an apparatus30 in FIGS. 2 and 3. Referring to FIG. 2, apparatus 30 comprises atubular column chamber 32 having an inlet end 34 and an outlet end 36.Outlet end 36 terminates proximate a plate 38. Plate 38 can comprise awindow configured to enable light to pass through for spectroscopicmeasurement of materials eluting from column chamber 30. A matrixmaterial 40 forms a packed column 42 within column chamber 32. Packedcolumn 42 has a lateral periphery defined by tubular chamber 32. Packedcolumn 42 can be formed by flowing a slurry of matrix material 40 and acarrier fluid into column chamber 32. Outlet end 36 of column chamber 32is displaced from plate 38 by a distance “D” sufficient to enable thecarrier fluid to pass between column chamber 32 and plate 38. However,the distance is less than an average width of matrix material 40.Accordingly, matrix material 40 is retained in column chamber 32 andstacks against plate 38 to form packed column 42.

[0012]FIG. 3 illustrates a method for removal of matrix material 40 frompacked column 42. Specifically, column chamber 32 is raised to enablematrix material 40 to pass beneath column chamber 32 and over plate 38.Subsequently, a fluid is flowed through chamber 32 to push matrixmaterial 40 out of column chamber 32.

[0013] System 30 is improved relative to other methods of packing andunpacking columns in that it can provide a quick method for releasingpacked column material from a column chamber, and can also provide aquick method for resetting the column chamber to be repacked with freshmatrix material. A difficulty with column system 30 is that it can beproblematic to move an entirety of column chamber 32 during transitionsbetween packing and unpacking operations. Further, precise tolerancesare needed to hold beads and may leak beads. Discharged beads canundesirably pass through a detector. It can become increasinglydifficult to move the entirety of column chamber 32 as a column-basedseparation is scaled up for larger operations. Accordingly, it isdesirable to develop alternative methods for conveniently packing andunpacking column chambers, wherein a column chamber is not moved intransitioning between packing and unpacking operations.

SUMMARY OF THE INVENTION

[0014] In one aspect, the invention encompasses a method of packing andunpacking a column chamber. A matrix material is packed within a columnchamber to form a packed column. After the packing, the matrix materialis unpacked from the column chamber without moving the column chamber.

[0015] In another aspect, the invention encompasses a method ofpurifying a component of a sample. A column chamber having an inlet endand an outlet end is provided. The outlet end terminates proximate botha first flow path and a second flow path. The first flow path isobstructed with a retaining material permeable to a first fluid andimpermeable to a matrix material. The second flow path is blocked by ablocking material that removably blocks flow of both the first fluid andthe column matrix material. A suspension of the first fluid and thematrix material is flowed into the column chamber and along the firstflow path to form a packed column of the matrix material within thecolumn chamber. The blocking material defines a portion of a peripheryof the packed column. The matrix material is configured to selectivelyretain a component of the sample. The sample flowed through the packedcolumn and along the first flow path to separate the component from therest of the sample. The blocking material is removed without moving thecolumn chamber. After removing the blocking material, a second fluid isflowed through the column chamber and along the second flow path toremove the matrix material from the column chamber.

[0016] In yet another aspect, the invention encompasses a system forcolumn-based separations. The system comprises a fluid passagewaycontaining a column chamber and a flow path in fluid communication withthe column chamber. The flow path is obstructed by a retaining materialpermeable to a carrier fluid and impermeable to a column matrix materialsuspended in the carrier fluid. The flow path extends through the columnchamber and through the retaining material. The flow path is configuredto form a packed column within the column chamber when a suspension ofthe fluid and the column matrix material flowed along the flow path. Thecolumn chamber defines a portion of a periphery configured to retain thepacked column. Another portion of the periphery is defined by a blockingmaterial that removably blocks flow of both the carrier fluid and thecolumn matrix material. The blocking material is spaced from the packedcolumn by a region configured to retain a fluid.

[0017] An advantage of the impermeable material is that the surface areaof the material in contact with fluid is always in contact with fluid.In other words, there is no material surface area that alternatelycontacts fluid then, say an interior chamber surface. This featureminimizes the potential of sample to sample contamination since a samplemay be completely washed through and not captured on an alternately orintermittently exposed surface. This is especially valuable for nucleicacid samples wherein one molecule of a previous nucleic acid sample canbe detected in a subsequent nucleic acid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Preferred embodiments of the invention are described below withreference to the following accompanying drawings.

[0019]FIG. 1 is a diagrammatic, cross-sectional view of a prior artcolumn construction.

[0020]FIG. 2 is a diagrammatic, cross-sectional view of a prior artsystem for packing and unpacking a column chamber. The system of FIG. 2is shown with the column chamber in a position for packing a matrixmaterial within the column chamber.

[0021]FIG. 3 is a view of the FIG. 2 system, with the column chambershown in a position for unpacking the column chamber.

[0022]FIG. 4 is a diagrammatic, cross-sectional view of a firstembodiment system of the present invention for packing and unpacking acolumn chamber. The system of FIG. 4 is shown in a position for packinga column chamber.

[0023]FIG. 5 is a view of the FIG. 4 system shown in a position forunpacking the column chamber.

[0024]FIG. 6 is a view of a second embodiment system of the presentinvention for packing and unpacking a column chamber. FIG. 6 is a viewof the system in a position for packing the column chamber.

[0025]FIG. 7 is a view of the FIG. 6 system shown in a position forunpacking a column chamber.

[0026]FIG. 8 is a schematic view of a first embodiment sample treatmentapparatus of the present invention. The apparatus of FIG. 8 incorporatesa third embodiment packing and unpacking system.

[0027]FIG. 9 is a schematic view of a second, embodiment sampletreatment apparatus of the present invention.

[0028]FIG. 10 is a schematic view of another embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In one aspect, the invention encompasses systems for column-basedseparations configured to pack and unpack column chambers without movingthe column chambers. Embodiments pertaining to this aspect of theinvention are described with reference to FIGS. 4-8, with FIGS. 4 and 5illustrating a first embodiment, FIGS. 6 and 7 illustrating a secondembodiment, and FIG. 8 illustrating a third embodiment.

[0030] Referring to FIGS. 4 and 5, a system 50 for column-basedseparations comprises a column chamber 52 having an inlet end 54 and anoutlet end 56. Column chamber 52 comprises a longitudinal axis “X”, andcan be formed of, for example, glass, plastic or metal. In the showncross-sectional sideview, column chamber 52 comprises a pair of opposingsidewalls 53 and 55. Although sidewalls 53 and 55 are shown asphysically separated in the shown cross-sectional view, it is to beunderstood that sidewalls 53 and 55 can be portions of a continuousperiphery. For instance, column chamber 52 can have a cylindrical shape,with sidewalls 53 and 55 forming portions of a continuous circularperiphery of the cylinder.

[0031] Outlet end 56 is obstructed with a retaining material 58 which ispermeable to a carrier fluid and impermeable to a column matrix materialsuspended in the carrier fluid. Retaining material 58 can comprise, forexample, a porous fritted glass and/or ceramic material. A first flowpath for fluids passing through column chamber 52 is defined to extendthrough retaining material 58. A first valve 64 is provided toselectively block flow along the first flow path. First valve 64 isshown in a “open” position in FIG. 4, enabling a fluid to flow along thefirst flow path.

[0032] System 50 further comprises a tube 60, which in the shownpreferred embodiment extends to the longitudinal axis “X” of columnchamber 52. It is further preferred that tube 60 extend at about a rightangle to the longitudinal axis “X”. A rotatable second valve 62 isprovided within tube 60 to regulate flow of material through tube 60.Second valve, 62 is configured to block flow of both fluids and matrixmaterial. FIG. 4 illustrates system 50 with second valve 62 in a“closed” position to prevent flow of fluid and matrix material throughtube 60. Tube 60 defines a second flow path for materials flowed intocolumn chamber 52, and in FIG. 4 such second flow path is illustrated asbeing blocked by closed valve 62.

[0033] In the shown cross-sectional view, sidewall 53 terminates in afluid tight seal at retaining material 58, and sidewall 55 terminates ata location elevationally displaced from retaining material 58. Secondvalve 62 is laterally displaced from column chamber 52 and can be usedto regulate flow of materials under sidewall 55. Specifically, whenvalve 62 is in a closed position, flow under sidewall 55 is prevented,and when valve 62 is in an open position, flow under sidewall 55 isenabled.

[0034] A support structure 66 is provided to support column chamber 52and tube 60. Support structure 66 can comprise, for example, a plasticmaterial molded to fit column chamber 52 and tube 60. Alternatively,support structure 60 can comprise, for example, a clamp.

[0035] A matrix material 68 is provided within column chamber 52. In theFIG. 4 orientation of system 50, wherein valve 62 is in a closedposition and valve 64 is in an open position, matrix material 68 ispacked within column chamber 52 to form a packed column 70. Such packingencompasses flowing a slurry of matrix material and carrier fluid intochamber 52 and along the first flow path though retaining material 58.The carrier fluid penetrates retaining material 58 and exists columnchamber 52, leaving matrix material 68 to stack against retainingmaterial 58 and form packed column 70. During packing of matrix material68, column chamber 52 defines a first portion 72 of a peripheryconfigured to retain packed column 70, and closed valve 62 defines asecond portion 74 of the periphery configured to retain packed column70. Specifically, closed valve 62 defines second portion 74 through aretaining fluid 76 that is within a fluid-filled space extending betweenvalve 62 and packed column 70. Retaining fluid 76 forms a boundary alongwhich matrix material 68 is packed to form packed column 70. In theshown preferred embodiment, retaining fluid 76 forms a barrier whichprecludes matrix material 68 from entering the fluid-filled spacebetween valve 62 and packed column 70 during formation of packed column70.

[0036] The retaining fluid 76 can be either a gas or a liquid, but ispreferably a liquid. Also, the carrier fluid is preferably a liquid. Inpreferred methods of the present invention, tube 60 and column chamber52 are initially filled with a first liquid prior to introduction of aslurry comprising a liquid carrier fluid and a matrix material withincolumn chamber 52. The liquid carrier fluid then flows through outlet56, while the first liquid remains within tube 60 to form a retainingfluid volume between valve 62 and packed column 70.

[0037] As packed column 70 is formed, a pressure may be exerted onretaining fluid 76 to elevate a pressure of retaining fluid 76 above oneatmosphere. This can occur, for example, if pressure is utilized toforce carrier fluid through retaining material 58.

[0038] Referring to FIG. 5, system 50 is illustrated with valve 64 in aclosed position and valve 62 in an open position. Opening valve 62unblocks flow of matrix material along tube 60, enabling discharge ofmatrix material 68 through tube 60. During discharge of matrix material68, a fluid, preferably a liquid, is flowed through column chamber 52and along the second flow path comprising tube 60 to flush matrixmaterial 68 from column chamber 52. The fluid flowed during discharge ofmatrix 68 can be referred to as a dislodging fluid, and can be the sameas one or both of the carrier fluid and the retaining fluid 76 (FIG. 4).

[0039] The system 50 described with reference to FIGS. 4 and 5 can beshifted from a packing mode to an unpacking mode by activating twovalves. Specifically, when valve 62 is in a closed position and valve 64is in an open position, system 50 is in a column chamber packing mode(FIG. 4), and when valve 62 is in an open position and valve 64 is in aclosed position, system 50 is in a column chamber unpacking mode (FIG.5). Thus, the embodiment of FIGS. 4 and 5 enables a system to be shiftedfrom a column chamber packing mode to a column chamber unpacking modewithout moving the column chamber.

[0040] Referring next to FIGS. 6 and 7, a second exemplary embodimentcolumn system 80 is illustrated. FIG. 6 illustrates column system 80 ina column chamber packing mode, and FIG. 7 illustrates column system 80in a column chamber unpacking mode. Referring first to FIG. 6, columnsystem 80 comprises a column chamber 82 having an inlet end 84 and anoutlet end 86. Outlet end 86 is obstructed with a retaining material 88permeable to a carrier fluid, and impermeable to a matrix material.Retaining material 88 can comprise, for example, a porous glass orceramic material.

[0041] System 80 further comprises a valve 102 having at least fourports and two positions. The four ports are labeled as 104, 106, 108,and 110. Port 110 is blocked with a blocking material 90. Ports 104 and108 are in fluid communication with column chamber 82, and comprise afirst flow path and a second flow path, respectively, from columnchamber 82. Port 106 is an unblocked outlet port which can lead to, forexample, a detector, a collection chamber, or additional equipment forprocessing substances eluted from packed column 92. Blocking material 90is configured to block flow of both column matrix material and carrierfluid, and can comprise, for example, a plastic plug threadably insertedinto port 110, or a suitable valve.

[0042] A packed column 92 of matrix material 94 is formed within columnchamber 80. Column chamber 80 defines a portion 96 of a periphery ofpacked column 92, and blocking material 90 defines another portion 98 ofthe periphery of packed column 92. Blocking material 90 is separatedfrom the periphery of packed column 92 by a fluid-filled region 100filled with a retaining fluid 101. Fluid-filled region 100 can comprise,for example, a plastic tubing.

[0043] In the shown embodiment, column chamber 82 comprises aright-angle bend 112 proximate retaining material 88, and accordinglyproximate an outlet extending through material 88. The portion 98 of theperiphery of packed column 92 defined by the retaining fluid 101 is atright-angle bend 112.

[0044] An alternative method of describing the apparatus of system 80 isto describe column chamber 82 as comprising a longitudinal sectionterminating at right-angle bend 112, and further comprising a fluidfilled region 100 comprising a tube at right angle bend 112. In theshown embodiment, the longitudinal section of column chamber 82,together 11 with right-angle bend 112, and the tubular portion of fluidfilled region 100 that joins with right-angle 112 define a “T” shape ina zone illustrated as zone “Z”.

[0045] Referring to FIG. 7, system 80 is shown with valve 102 moved intoa second position which couples port 104 with blocked port 110 and whichcouples port 108 with open port 106. The second position of valve 102thus blocks the first flow path through retaining material 88 and opensthe second flow path adjacent column chamber 82 to enable discharge ofmatrix material 94 from column chamber 82.

[0046] The above-discussed embodiments of FIGS. 4-7 can enable a columnchamber to be packed and unpacked without moving the column chamber. Theembodiments utilize a blocking material (either valve 62 or material 90)to removably block flow of fluids and column matrix material from acolumn chamber. When flow is blocked, the column chamber can be packedwith a matrix material, and when the flow is unblocked the columnchamber can be unpacked.

[0047] The column chambers of FIGS. 4-7 are preferably packed andunpacked with matrix material while flowing fluids through the columnchambers in an identical flow direction during both the packing andunpacking operations. Specifically, all, or at least most, of the matrixmaterial is preferably expelled from a column chamber along an identicalflow direction as was utilized to pack the column chamber. However, itis to be understood that the invention also encompasses embodiments inwhich flow of fluid through a column chamber is reversed during apacking or unpacking operation. Preferably, the flow during a packingoperation will not be reversed, but will instead be continuous in a flowdirection from an inlet of the column chamber through a retainingmaterial (such as retaining material 88 of FIGS. 6 and 7). Alsopreferably, a flow of fluid through a column chamber will bepredominately in a single direction from an inlet of the column throughan outlet of the column during an unpacking operation. However, theunpacking operation can preferably also comprise some sporadic instanceswherein flow is reversed (i.e. to go from an outlet to an inlet) toassist in dislodging matrix material from within column chamber 82, andto assist in removing matrix material embedded within a porous retainingmaterial (such as retaining material 88 of FIGS. 6 and 7) that couldotherwise potentially clog the retaining material.

[0048] Embodiments of the present invention can be operated with a vastnumber of matrix materials, as will be appreciated by persons ofordinary skill in the art. Example matrix materials are the Sr-resin,TRU-resin, and TEVA-resin described above in the “Background” section ofthis disclosure, which can be utilized for separating compoundscomprising radioactive atoms from other materials in a sample.Additional example matrix materials including but not limited to glass,sepharose, polystyrene, Tepnel, Qiagen, zirconium, hydroxyapatite,POROS, PEG-PS, and PS the last three of which are made by PerSeptive arematerials, suitable for separating biological materials. Certain matrixmaterials are materials for separating nucleotide fragments (e.g.,nucleic acid, DNA, RNA or combinations thereof) based upon a sequence ofthe fragments, such as, for example, the Tepnel micro-beads discussedabove in the “Background” section of this disclosure. Biologicalmaterials include but are not limited to viruses, cells for exampleprokaryote, eukaryote and combinations thereof.

[0049] Column systems of the present invention can be incorporated intomethods for purifying components of samples. Example purificationapparatuses are shown in FIGS. 8 and 9, with FIG. 8 showing a firstembodiment apparatus and FIG. 9 showing a second embodiment apparatus.

[0050] Referring to FIG. 8, a purification apparatus 120 comprises apump 122 (for example, a syringe pump), valves 124, 126, 128 and 130,and a column chamber 132. Exemplary valves 128 and 130 are 4 port, 2position diverter valves such as may be obtained from, for example,Valco as Cheminert valves. Valve 130 comprises ports 140, 142, 144 and146, with port 146 obstructed by a filter 148. Filter 148 can comprise,for example, a 25 μm poresize polypropylene column end frit with a 5 mmdiameter, and can be placed into a port of a 2 position diverter valvewith a flat-bottom chromatographic fitting. Column chamber 132 is packedby flowing a slurry of carrier fluid and matrix material into columnchamber 132 while a fluid flow is coupled through ports 144 and 146.Filter 148 is permeable to the carrier fluid and impermeable to thematrix material. Column chamber 132 can be subsequently unpacked byactivating valve 130 to couple a fluid flow through ports 144 and 142.Port 142 is preferably an open port sized to permit flow of a slurrycomprising matrix material through port 142. Column chamber 132 andvalve 130 together comprise a third embodiment column system of thepresent invention that can be packed and unpacked without moving acolumn chamber.

[0051] Valve 128 comprises ports 150, 152, 154 and 156. Port 152 canlead to, for example, a waste reservoir (labeled “W”). Port 150 ispreferably in fluid communication with a wide dimension tubing 151, suchas, for example, tubing having an internal diameter of 1.6 mm, to enablea slurry comprising matrix material and carrier solution to easily flowthrough the wide bore tubing during packing of column 32. Port 154 ispreferably in fluid communication with tubing 155 having a relativelynarrow bore, such as, for example, an internal diameter of 0.8 mm. Suchnarrow bore tubing enables a sample to be flowed onto column 32 withless dilution of the sample than would occur with a wider diametertubing. In operation, a fluid flow is coupled through ports 150 and 156during packing of column 32, and a fluid flow is coupled through ports154 and 156 during loading of a sample onto column 152. Either of ports154 or 150 can be coupled to port 156 if during elution of sample frommatrix material of column 132. A sample eluted from the matrix materialof column 132 can be passed through filter 148 to a detector 170downstream of port 146. Detector 170 can comprise, for example, apolymerase chain reaction (PCR) machine.

[0052] Valves 126 and 124 are utilized for providing reagents, samples,and slurries to column 132. Preferably, if a slurry is to be provided tocolumn 132, it is first flowed into tubing segment 172, that can be anyshape. Tubing segment 172 is preferably coiled as shown to save space.The slurry subsequently flow from tubing segment 172 to column 132. Byflowing the slurry to tubing segment 172, the slurry can be providedwithin apparatus 120 without introduction of gas bubbles into the tubingof the apparatus. Specifically, tubing segment 172 can be filled with aliquid (preferably a liquid inert to reaction with the slurry) prior toinjection of slurry into holding coil 172. The slurry can then beinjected into tubing segment 172 to displace the liquid. To preventmixing of the injected slurry with the displaced liquid, a small airbubble (about 100 microliters) can be introduced into tubing segment 172prior to injecting the slurry.

[0053] Each of valves 126, 128 and 130 preferably comprises a port goingto a waste material reservoir “W”. Example dimensions of various itemsof apparatus 120 are as follows: column 132 can comprise a volume ofabout 250 μL, holding coil 172 can comprise a void volume of about 12mL, and syringe pump 122 can comprise a displacement of about 10 mL. Atypical time for packing a 250 microliter column 132 is less than fourand one-half minutes. The holding coil, column chamber and tubing ofapparatus 120 can comprise, for example, TEFLON™, with the holding coiland tubing preferably comprising FEP (fluorinated ethylenepropylene)-TEFLON and the column chamber preferably comprising PTFE(polytetrafluoroethylene)-TEFLON. Example conditions for utilizingapparatus 120 are described in Tables 1-5. TABLE 1 Automated ProtocolFor Renewable Sorbent Column Packing (Described With Reference To TheApparatus Of FIG. 8) Step# Event (Flow Rate)  1. switch two-positionvalves 128 and 130 to positions 2,   and 1, respectively  2. aspirate100 μL of air into the holding coil (15 mL/min)  3. aspirate 635 μL ofcarrier into a syringe (35 mL/min)  4. aspirate 700 μL of sorbent slurryinto the holding coil (3 mL/min)  5. dispense 700 μL of sorbent slurryto renewable column via a packing line (3 mL/min)  6. repeat steps 4 and5 as necessary^(a)  7. dispense 635 μL to the packing line (3 mL/min).Pause   12 seconds.  8. switch two-position valve 128 to position 1.  9.aspirate 100 μL of air into the holding coil (15 mL/min) 10. aspirate750 μL of carrier into a syringe (35 mL/min) 11. dispense 700 μL throughthe slurry delivery line to the waste line (6 mL/min) 12. aspirate 100μL of air into the holding coil (15 mL/min) 13. aspirate 750 μL of 50%ethanol solution into the holding coil (15 mL/min) 14. aspirate 1.8 mLof carrier into the syringe (35 mL/min) 15. empty syringe through theslurry delivery line to waste (10 mL/min)

[0054] TABLE 2 Automated Protocol for Sorbent Bed Disposal (DescribedWith Reference To The Apparatus Of FIG. 8) Step# Event (Flow Rate)  1.switch two-position valves 128 and 130 to positions 2  2. aspirate 350μL through the column body and slurry delivery line (10 mL/min)  3.aspirate 100 μL of air into holding coil (15 mL/min)  4. aspirate 4.25mL of carrier into syringe (35 mL/min)  5. switch two-way valve 130 toposition 1  6. aspirate 100 μL through the column body and packing line(10 mL/min)  7. switch two-way valve 130 to position 2  8. dispense 900μL through the column body to waste (10 mL/min)  9. switch two-way valve130 to position 1 10. aspirate 100 μL through the column body (10mL/min) 11. switch two-way valve 130 to position 2 12. aspirate 100 μLof air into the holding coil (15 mL/min) 13. dispense 1.5 mL through theslurry line and column body to waste (10 mL/min) 14. switch two-wayvalve 130 to position 1 15. aspirate 100 μL of air into the holding coil(15 mL/min) 16. aspirate 1.2 mL of 50% ethanol solution into the holdingcoil (10 mL/min) 17. dispense 2.6 mL through the slurry delivery lineand column body to waste (10 mL/min)

[0055] TABLE 3 Automated Reagent Delivery Sequence for ⁹⁰SrSeparation^(a,b) Step# Description: Reagent (Flow Rate) 1. pack Sr-resincolumn 2. condition column: 1.5 mL 8 M HNO₃-0.12 M HF (1 mL/min) 3. loadsample/wash column: 150 μL 8 M HNO₃- 0.12 M HF/100 μL sample/6 mL 8 MHNO₃-0.12 M HF (0.5 mL/min or 1 mL/min) 4. elute Sr: 5 mL 0.05 MHNO₃-0.12 M HF (0.5 mL/min) 5. expel Sr-resin sorbent

[0056] TABLE 4 Automated Reagent Delivery Sequence for Am Separation^(a)Step# Description: Reagent (Flow Rate) 1. pack TRU-resin column 2.condition column; 1.5 mL 2 M HNO₃ (1.0 mL/min) 3. load sample/washcolumn: 100 μL sample/6 mL 2 M HNO₃ (1.0 mL/min) 4. elute Am: 4 mL 3 MHCl (1.0 mL/min) 5. elute actinides: 4 mL 0.1 M ammonium bioxalate (1mL/min) 6. expel TRU-resin sorbent

[0057] TABLE 5 Automated Reagent Delivery Sequent for ⁹⁹Tc SeparationStep# Description: Reagent (Flow Rate) 1. pack TEVA-resin column 2.condition column: 1.5 mL 0.5 M HNO₃-12 M HF (0.5 mL/min) 3. load sample:400 μL sample solution (0.5 mL/min) 4. wash column: 5 mL 0.5 M HNO₃-12 MHF (0.5 mL/min) 5. wash column: 1.5 mL deionized water (0.5 mL/min) 6.expel TEVA-resin sorbent and collect in vial

[0058] Referring next to FIG. 9, a second embodiment sample purificationapparatus 200 comprises a pump 202 (such as a shown peristaltic pump), acolumn system 204, and valves 206 and 208. Column-system 204 cancomprise, for example, one of the above-discussed first, second or thirdembodiment column systems of the present invention. Column system 204 isshown with a single outlet 210 which can, for example, correspond to asingle outlet from outlet port 106 of system 80 (FIGS. 7 and 8), or cancorrespond to a joined outlet formed from joining the first and secondflow paths of system 50 (FIG. 4) in an embodiment of system 50 which isnot shown.

[0059] Apparatus 200 further comprises a holding coil 212 configured forholding either a sample which is to be separated with column system 204,or a slurry which is to be utilized for forming a packed column.Apparatus 200 can have particular application for separating componentsof biological samples. For instance, a desired component of a biologicalsample will frequently be present to a small concentration in a largesample. Apparatus 200 permits the sample to be repeatedly cycled acrosscolumn system 204, which can increase an amount of a biologicalcomponent ultimately bound to a packed column relative to an amountwhich would be bound if a sample were not cycled multiple times acrosscolumn system 204.

EXAMPLE 1

[0060] Experiments were conducted to demonstrate radiologicalseparations using the apparatus and method of the present invention. Theapparatus used was as described above as the third embodiment. Automatedprotocols for column packing and unpacking are described above in Table1 and Table 2. In an example method the column system in FIG. 8 was usedto perform analytical separation and determination of ⁹⁰Sr in nuclearwaste samples using Sr-resin. An on-line liquid scintillation detectorwas used to observe eluting peaks. The sample was loaded on the columnin strong nitric acid solution (>3 M concentration), where Sr ions werestrongly and nearly selectively retained. The matrix and mostinterfering radioactive ions, including the ⁹⁰Y daughter, showed noretention and were removed with a strong nitric acid wash. Strontium wasthen eluted using a dilute nitric acid solution (ca. 0.05 M). Theprotocol for implementing the separation is shown above in Table 3.Reagent solution of 8 M HNO₃-0.12 M HF was used as a column wash toensure removal of the tetravalent actinides which were coretained with⁹⁰Sr.

[0061] The experimental procedure listed in Table 3 was applied to ahigh activity ⁹⁰Sr/⁹⁰Y standard (2.14×10⁵ ⁹⁰Sr dpm/mL in 2 M HNO₃) intriplicate, the separation flow rate was 0.5 mL/min. For triplicate runson standards, both net peak area and peak maximum counts werereproducible within a 2 s counting error (3% and 9% respectively). Theseparated Sr fractions from the standard runs were collected and countedoff-line to estimate the on-line detection efficiency (E_(d)) andseparation recovery (E_(s)). The ⁹⁰Sr separation recovery, E_(s), was92±2%, and the on-line detection efficiency, E_(d), was 62±3%. Reuse ofSr-resin generally requires additional column wash steps in order toreduce strontium carryover into the subsequent analysis. A reagent blankrun using a conventional chromatographic column performed immediatelyafter the separation of a high activity ⁹⁰Sr standard indicatedcarryover of ˜7%. However, no carryover was evident if the reagent blankrun following the analysis of the high-activity standard was performedon the automatically repacked column. No carryover was detected using amore sensitive off-line liquid scintillation analysis of a collected Srfraction (less than 0.07% carry-over).

[0062] Consequently, one of the primary benefits, of using renewablecolumn technique demonstrated is that analyte carryover into succeedinganalyses due to retention on column material is eliminated. Lengthycolumn clean-up steps and blank runs, which are sometimes required whenreusing sorbent extraction columns, were unnecessary when using therenewable column.

[0063] The renewable column apparatus shown in FIG. 8 was applied toseparation of ²⁴¹Am from nuclear waste sample for determination byalpha-spectroscopy using TRU-resin. Determination of ²⁴¹Am in nuclearwaste samples using alpha-spectroscopy requires that Am is separatedfrom the stable matrix, highly radioactive fission products, andpotential radioactive interferences. (e.g., ²³⁸Pu). The procedure givenin Table 4 was applied to a dissolved vitrified glass nuclear wastesample prepared in 2 M HNO₃-0.05 M NaNO₂. The sample was spiked with5.0×10⁵ dpm/mL of ²⁴¹Am and ²³⁹Pu. On-line radioactivity detection wasused to monitor the separation. The ammonium bioxalate elution step isrequired if the TRU-resin column is to be reused for subsequent Amseparations. However, using the renewable column technique, the sorbentcolumn can be automatically repacked after the Am elution step. In thiscase, there was no need to elute the actinides still present on thecolumn (Table 4, step 5). Additional column wash steps to reducepotential carryover into subsequent analysis were also eliminated. Theseparation time was reduced and mixed waste generation was minimized.This separation approach was applied towards the analysis of nuclearwaste samples with off-line detection by alpha spectrometry. Analyticalresults were in close agreement with those obtained by standard manualprotocols.

[0064] Renewablc column technique was used to for separation andanalysis of ⁹⁹Tc in nuclear waste samples using TEVA-resin. Theautomated protocol listed above in Table 5 was developed to pack andcondition TEVA-resin column, load the sample, and perform the columnwash that removes stable matrix and radioactive interferences (e.g.,¹³⁷Cs and ⁹⁰Sr/⁹⁰Y). Eluent comprising 0.05 M HNO₃-0.12 M HF reagent wasused as a wash to eliminate retention of the teravalent Pu. The samereagent was used to perform Sr elution as described earlier. Residualnitric acid present on the column after the column wash step was removedusing 1.5 mL of water (Table 5, step 5). After the sample load andcolumn wash steps (Table 5), the TEVA-resin sorbent was expelled fromthe system. The sorbent slurry in water (˜3 mL) was collected into avial, mixed with 15 mL of scintillation cocktail and analyzed off-lineby liquid scintillation spectrometry.

[0065] The following samples (1 mL aliquots of 0.1 M HNO₃ solutions)were analyzed using the SI-RSC separation format: 1) reagent blank, 2)tank waste sample; 3) tank waste sample spiked with 2300 dpm/mL⁹⁹Tc(VII), and 4) tank waste sample spiked with 4350 dpm/mL of⁹⁹Tc(VII). Blank subtracted detector count rate (cpm) plotted for thesample and two spiked samples against the added standard activity (dpm)gave a straight line (cpm=0.937 dpm+358, R=1.000). The sample ⁹⁹Tcactivity was determined from the x-axis intercept of the standardaddition plot. The detection efficiency for the heterogeneous TEVA-resinslurry/scintillation cocktail samples was estimated to be 95±4%. Theseparation recovery was quantitative (99±5%). The analysis result (±2 s,corrected for dilutions) for a tank waste sample obtained using theSI-RSC technique was 1240±163 dpm/mL. This is in satisfactory agreementwith the value of 1121±146 dpm/mL determined by standard analysisprocedures. These results demonstrated a unique capability of thedescribed technique to perform automated separations that requirerecovery of spent sorbent for subsequent analytical steps.

EXAMPLE 2

[0066] DNA Purification from Soil Extracts

[0067] DNA Extraction Procedure

[0068] This example demonstrates the utility of the renewablemicrocolumn for DNA purification from complex samples. This exampleincludes 60 m particles with DNA binding sites, however any DNA bindingparticles ranging in size from about 10 μm to 200 μm could be used withthis procedure for. DNA purification. A schematic of the system used forthese experiments is shown in FIG. 10. The microcolumn 50 was machinedfrom Plexiglass to include a 1.8 mm diameter column 52. The columnincluded a PEEK frit 58 with a 10 μm pore size (Upchurch). A syringepump 122 (Cavro) was used for single pass experiments, and an Eldexreciprocating pump 1002 was added for recirculating the sample throughthe column 52. An eight port valve 1000 was used to direct flow ofsolutions. A typical, aenalytical protocol is outlined in Table E2-1.TABLE E2-1 Procedural Solution Step Composition Solution volume FlowrateAdd 15 mg/ml To produce 7 μl   3 μl/s Column Tepnel 1392r packed bedbeads in 0.3 M volume NaCl Inject 1 ng or 100 ng  200 μl 0.9 μl/s SampleGeobacter chapellii DNA in 0.3 M NaCl (or soil extract) (DNAconcentration is 8.3 × 10⁻¹³ M or 8.3 × 10⁻¹⁵ M) Rinse 0.3 M NaCl  80 μl  3 μl/s Column Wash 0.5X SSC 1700 μl   3 μl/s Column (0.075 M NaCl,0.0075 M NaCitrate) Elute Water  100 μl 0.3 μl/s DNA

[0069] Summary of the Automated DNA Sample Processing Procedure Used forExtraction of Geobacter chapellii DNA Spiked into Clean Solutions andCrude Soil Extracts.

[0070] Tepnel microbeads derivatized with universal 1392roligonucleotide were used for the purification. The flowrates for sampleinjection and DNA elution are average flowrates produced by alternatingflow at 3 μl/s and stopped flow. After the DNA is eluted, the column isautomated flushed from the system.

[0071] The microbeads for DNA purification were obtained from TepnelLife Sciences (Cheshire, England) and included universal 16S rDNAoligonucleotide 1392r with a dT₈ linker covalently attached to 60 μmmicrobeads. The binding capacity was estimated to be 2 pmol mg⁻¹ (orcm²) beads (1.27×10¹² capture probes mg-⁻¹) based upon a competitivehot/cold assay using complementary oligonucleotides (Tepnel).Underivatized beads were also obtained to test for non-specific bindingof nucleic acids. Before experimenting with Tepnel reagents in thefluidic system, we performed batch capture experiments using bothunderivatized and 1392r-derivatized microbeads.

[0072] For each nucleic acid size and quantity, automated captureexperiments were performed with 200 μl blank extract (no DNA), purifiedG. chapellii genomic DNA in 0.3M NaCl, and at least two replicatecaptures of G. chapellii genomic DNA in 200 μl soil extract. The sampleprocessing times for the primary separation events are shown in TableE2-2. The nucleic acid capture program was initiated by delivering theaffinity matrix from a stirred slurry (15 mg ml⁻¹ in 0.3 M NaCl) to therenewable column, resulting in a 7 μl bed volume (1.6 mm ID×3.5 mmcolumn height) containing approximately 3.7 mg bead material, 3.5 cm²surface area and 4.4×10¹² capture probes. TABLE E2-2 DNA ExtractionSummary: Sample Processing Time 1 pass 10 pass batch automated automatedmanual sample contact   3 min 6 sec   11 min 7 sec 240 min time (0.93μl/s) (3 μl/s) (flowrate) total hybridization 3.5 min   19 min 240 mintime^(a) rinse time   9 min   9 min  10 min^(b) (flowrate) (3 μl/s) (3μl/s) elution time 5.5 min  5.5 min  10 min^(b) total processing  18 min33.5 min 260 min time #Processing times in the automated system were notoptimized for speed, and therefore do not necessarily reflect a lowerlimit on processing speed within the system and for other samplematrices.

[0073] Nucleic acid extracts (200 μl)-were heat denatured at 100° C. for10 min., quick-chilled on ice and perfused over the microcolumn at 0.9μl sec⁻¹ for a total contact time of approximately 3 min. The unboundflow-through was collected for subsequent analysis. Beads were washed byperfusing with 80 μl. 0.3M NaCl to remove the nucleic acid extract, andbound nucleic acids were washed in 1.7 ml 0.5×SSC (3 μl sec⁻¹), andhybridized target was eluted with 100 μl water at room temperature witha 5.5 min contact time. The total sample processing time from injectionto elution was 18 minutes. The experimental conditions were identicalfor the 10 pass sample recirculation experiments, except the extract andhybridization solutions were recirculated 10 times over the column at aflow rate of 3 μl s⁻¹, resulting in a sample contact time ofapproximately 11 minutes instead of 3 minutes, and a total hybridizationtime of 19 minutes (Table E2-2). Eluted nucleic acids were lyophilizedto dryness and resuspended in 20 μl water prior to PCR detection.Between captures, the flow system was washed extensively with a sequenceof 0.16% Roccal microbial disinfectant, 10% bleach, and water.

[0074] PCR Amplification and Measurement of Capture Efficiency

[0075]Geobacter 16S rDNA and total eubacterial 16S rDNA were detectedand enumerated using dilution-to-extinction PCR to estimate captureefficiency and, for captures in soil extracts, to provide a functionalassay for DNA purity that cannot be obtained by scintillation countingof radiolabeled DNA. Estimates of capture efficiency were calculated by(PCR detection limit)×(dilution factor)×(conversion factor to accountfor entire eluant). For example, with a 64 copy detection limit, 5³dilution to the last positive PCR signal, and a 2 μl DNA input into the5-fold dilution series (10% of the total recovered 0.5 DNA), 8×10⁴copies of DNA were recovered. At 1 ng input DNA (1×10⁶ copies of target,assuming one 16S rDNA copy per genome), the capture efficiency istherefore (8×10⁴/1×10⁶)×100, or 8%. Capture efficiencies calculated inthis manner are conservative estimates and underestimate the actualcapture efficiency. That is, the true extinction point of the PCR liesbetween the last positive signal and the next dilution in the 5-foldseries. Further, the positive control used to calibrate the enumerationswas non-fragmented, highly purified DNA rather than sheared DNA isolatedfrom a soil extract (which may amplify with lower efficiency than thestandard). These variables bias the calculation of capture efficiencydownward, such that the actual capture efficiency calculated in theexample above is >8% but <40%.

[0076] Purified DNA was serial diluted in a 5-fold series immediatelyprior to PCR. PCR primers were synthesized by Keystone Labs (Camarillo,Calif.): Gbc.1300f and Gbc.1400r; S-d401F-20 and S-d683aR-20; universaleubacterial primers fD1 and rP2. For both sets of Geobacter-specificprimers, PCR reactions were carried out in 25 μl total volume, utilizinga Perkin-Elmer (Foster City, Calif.) 9600 thermal cycler and 0.2 mlthin-walled reaction tubes. Final reaction conditions were 2 μl purifiedDNA (or dilutions thereof), 10 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl₂,200 μM each dNTP, 0.2/M each primer, and 0.625 U Taq polymerase (PerkinElmer) which had been pretreated with TaqStart™ antibody at 0.5× therecommended concentration (Sigma, St. Louis, Mo.). Assembled reactionswere heated to 80° C. for 5 min (hot start) and amplifications wereconducted by performing 5 cycles at 94° C. for 40 s, 60° C. for 10 s,72° C. for 75 sec followed by 40 cycles at 94° C. for 12 s, 65° C. for10 s, 72° C. for 80 s with a 2 s extension per cycle. A final 20 min,72° C. extension was performed before chilling reactions to 4° C.Control reactions included no template, solution blank and system washes(pre-and post-capture), affinity-purified nucleic acids (i.e. systemeluant) spiked with 250 fg G. chapellii genomic DNA, and a dilutionseries of G. chapellii genomic DNA. PCR conditions for universaleubacterial primers fD1/rP2 were essentially identical, except weutilized 1.5 mM MgCl₂ and a thermal profile consisting of 5 cycles at94° C. for 40 s, 55° C. for 10 s, 72° C. for 75 sec. 30 cycles at 94° C.for 12 s, 65° C. for 10 s, 72° C. for 80 s with a 2 s extension percycle, and a 20 min, 72° C. final extension.

[0077] DNA Purification Results

[0078] Relative to batch capture protocols, the automated capture wasfaster (18 versus 260 minutes total processing time (Table E2-2) andresulted in an extraction efficiency that was equivalent to or betterthan that obtained in batch solution using the same reagents. The bestbatch capture efficiency was only 2% using 100 ng of 4-10 Kb DNA and ahybridization time of 240 minutes, and ≦0.4% for a 30 minutehybridization. Automated capture of the same DNA size fraction andconcentration resulted in an extraction efficiency of 6.25%, with asingle pass through the column and a hybridization time of 3.5 minutes.

[0079] DNA capture with one pass over the renewable affinity columnvaried appreciably as a function of target size and absolute targetconcentration as seen in Table E2-3. At 1 ng of input genomic DNA (ca.10⁶ copies or 1.7 attamoles target), capture efficiency declined withthe smaller DNA fragment sizes; for 100 ng genomic DNA inputs (ca. 10⁸copies or 170 attamoles target), capture efficiencies were relativelyconstant and represented the highest observed capture efficiencies(6-30%). PCR analysis of the sample flow-through, column eluent andspent microbeads showed that the, majority of DNA applied to the columncould be detected in the flow-through fraction, indicating thatsignificant amounts of DNA were not adsorbed by the fluidics system(tubing, valves) and detectable target DNA was not retained on themicrobeads after elution. Automated capture of 1 Kb sheared DNA after 10passes (ca. 19 min. hybridization time) over the microcolumn showed noimprovement relative to single-pass experiments, suggesting that thecapture efficiency was not limited by column contact time alone. TABLEE2-3 Genomic DNA added 4-10 Kbp 1 Kbp 0.5 Kbp No DNA Sheared ShearedSheared added  1 ng Clean   8% 1.6%  0.3% 0% Background Soil 0.3% 0.3% 0.3% 0% Background (0.04%) 100 ng Clean   6%  31%    6% 0% BackgroundSoil 0.3% 0.3% 0.002% 0% Background (0.1%) (0.1%) (1%) (0.3%) 

[0080] Capture efficiency of Geobacter chapellii 16S rDNA with theTepnel-1392r reagent and one pass through the renewable affinity column.Each value is the average of at least two capture experiments. 1 ng ofG. chapellii genomic DNA=10⁶ cell equivalents (1 fg cell⁻¹) or 10⁶copies of target assuming 1 copy per cell. The estimated DNA content ofthe soil extract background was 3 μg per capture, or ca. 3×10⁹competitive DNA targets assuming 5 fg cell⁻¹ and one 16S rDNA target perfg. Values in parentheses indicate capture efficiency of total 16S rDNAtarget based on dilution-to-extinction PCR using universal 16S rDNAprimers fd1/rP2. Further, the maximal amount of target DNA recoveredfrom 1 ng genomic DNA captures was ca. 80 pg (ca. 8×10⁴ copies), whereasthe amount of target DNA recovered from the 100 ng genomic captures was6-30 ng (6×10⁶−3×10⁷ copies). Therefore, the limited capture of 1 ngtarget at all size ranges was not due to surface saturation of availablebinding sites, suggesting that kinetic and/or thermodynamic effectslimited nucleic acid capture at the lower target concentrations.

[0081] The capture efficiency for competitive eubacterial 16S RDNA fromunspiked soil extract was 0.3%, similar to the capture efficiency ofGeobacter genomic DNA targets that were also spiked into the soilbackground (Table E2-3). At both 1 ng and 100 ng of 4-10 Kb and 1 Kb DNAinputs into the soil background, the G. chapellii 16S rDNA specificcapture and total eubacterial 16S rDNA capture were constant and ofsimilar magnitude (0.3% capture efficiency), even though 100 ng of G.chapellii target constituted ca. 3% of the total 16S rDNA and 1 ngrepresented only 0.03% of total 16S rDNA target. These results indicatethat the spiked DNA (up to 10⁸ additional targets) did not appreciablychange the availability or binding efficiency of Geobacter targetsrelative to total, eubacterial, competitive 16S rDNA targets, and thathumic acids did not bias the affinity binding for or against the addedGeobacter DNA relative to indigenous 16S rDNA. In addition, theseresults indicate that the competitive DNA background did not bias theaffinity capture system for or against low-copy genomic DNA targets(Geobacter 16S rDNA) in solution. However, the binding of competitive16S rDNA sequences to the 1392r microbeads or non-specific binding ofhumic acids precluded more efficient binding of Geobacter target DNA,since purified Geobacter DNA was captured with up to 30% efficiency (at100 ng) whereas Geobacter targets spiked into a soil background (at 100ng) were captured with 0.3% efficiency.

[0082] These results demonstrate that the renewable microcolumn can beused to automate the purification of nucleic acids contained in complexsamples such as a crude soil extract. The protocol can include passingthe sample only one time over the column or recirculating the samplemany times over the column. The extraction efficiency obtained using theautomated system was equal to or better than the manual extractionefficiency using the same reagent. DNA (1 ng or 100 ng) in a soilextract was detectable by PCR after a single pass over the microcolumnusing the automated system and only 18 minutes total processing time.This processing time was not optimized for speed, so does notnecessarily reflect a lower limit on processing speed within the systemand for other sample matrices. Although the capture efficiency usingthis reagent was not high, the purified DNA sample does not inhibit PCRdetection. Without DNA purification prior to PCR, the DNA in the soilextract cannot be detected using PCR because of PCR inhibition byconstituents of the soil extract. The automated renewable microcolumnsystem could also be used with other purification resins that: bind DNA,or resins that bind other biomolecules, cells, or chemicals.

[0083] Closure

[0084] While a preferred embodiment of the present invention has beenshown and described, it will be apparent to those skilled in the artthat many changes and modifications may be made without departing fromthe invention in its broader aspects. The appended claims are thereforeintended to cover all such changes and modifications as fall within thetrue spirit and scope of the invention.

1. A method of packing and unpacking a column chamber, comprising:flowing a mixture of matrix material and fluid into a column chamber andforming a packed column from the matrix material, said chamber having afirst port having a retaining material permeable to said fluid andimpermeable to said matrix material; and closing said first port andopening a second port that is permeable to both the fluid and the matrixmaterial thereby unpacking the matrix material from the column chamberwithout moving the column chamber.
 2. The method of claim 1 wherein theunpacking removes substantially all of the matrix material from thecolumn chamber.
 3. The method of claim 2 wherein all of the matrixmaterial is removed from the column chamber.
 4. The method of claim 1wherein a first fluid is flowed into the column chamber during packingand a second fluid is flowed into the column chamber during unpacking,the second fluid being different from the first fluid.
 5. The method ofclaim 1 wherein said first and second ports are on a valve having atleast 2 positions, the method further comprising: flowing the mixture offluid and matrix material into the column chamber and along a flow pathobstructed by the retaining material during the forming the packedcolumn, the valve being in a first of the at least 2 positions duringthe forming the packed column and preventing a flow of fluid through thesecond port, a retaining fluid extending between the valve and thematrix material during the packing, the retaining fluid comprising aboundary at a periphery of the packed column; and after the packing,changing the valve position to a second of the at least 2 positions andflowing a dislodging fluid into the column chamber and through thevalve, the second of the at least 2 valve positions directing thedislodging fluid along a second flow path which extends through thesecond port and which is not obstructed by the retaining material tounpack the column.
 6. The method of claim 5 wherein said valve has atleast 4 ports.
 7. The method of claim 6 wherein the retaining materialis between the outlet of the column chamber and one of the ports, andanother of the ports is blocked with a material impermeable to bothliquid and the matrix material.
 8. A method of forming a packed column,comprising: providing a column chamber, the column chamber having aninlet end and an outlet end, the outlet end terminating proximate both afirst flow path and a second flow path, the first flow path obstructedwith a porous retaining material permeable to a first fluid andimpermeable to a matrix material, and the second flow path blocked; andflowing a mixture of the first fluid and the matrix material into thecolumn chamber and along the first flow path to pack the matrix materialwithin the column chamber.
 9. The method of claim 8 further comprising,after packing the matrix material in the column chamber, opening thesecond flow path and flowing a second fluid through the column chamberthereby unpacking the matrix material from the column chamber.
 10. Themethod of claim 9 wherein the first fluid and the second fluid are thesame.
 11. The method of claim 9 wherein the second flow path is blockedwith a retaining fluid.
 12. The method of claim 11 wherein the firstfluid, the second fluid, and the retaining fluid are the same.
 13. Themethod of claim 11 further comprising: after packing the matrix materialin the column chamber, removing the retaining fluid and flowing thesecond fluid through the column chamber and along the second flow pathto unpack the matrix material from the column chamber; and reversing aflow of the second fluid along the second flow path while unpacking thematrix material from the column chamber.
 14. The method of claim 8wherein: said column chamber in cross-sectional sideview comprises apair of opposing sidewalls, one of the opposing sidewalls terminating ina fluid-tight seal at the retaining material, the other of the sidewallsterminating at a location elevationally displaced from the retainingmaterial; providing a valve laterally displaced from the column chamber,the valve having a closed position and an open position, the valve inthe closed position substantially preventing fluid flow under the otherof the sidewalls, the valve in the open position enabling fluid flowunder the other of the sidewalls; positioning the valve in the closedposition and flowing a mixture of the first fluid and the column matrixmaterial into the column chamber, the first fluid flowing through theretaining material and the column matrix material being retained by theretaining material to form a packed column within the column chamber;and after forming the packed column, positioning the valve in the openposition and flowing a dislodging fluid into the column and under theother of the sidewalls to flush the matrix material under the other ofthe sidewalls and thereby unpack the column from the column chamber. 15.A method of forming a packed column, comprising: providing a columnchamber having an outlet, the column chamber defining a portion of aperiphery configured to retain a column matrix material, another portionof the periphery being defined by a retaining fluid; obstructing theoutlet of the column chamber with a porous retaining material permeableto a carrier fluid and impermeable to a column matrix material suspendedin the carrier fluid; and flowing a suspension of the carrier fluid andthe column matrix material into the column chamber to form a packedcolumn within the column chamber.
 16. The method of claim 15 furthercomprising, after forming the packed column, removing the retainingfluid and flowing a dislodging fluid through the column chamber tounpack the column.
 17. The method of claim 15 wherein the carrier fluid,the dislodging fluid, and the retaining fluid are the same in chemicalcomposition.
 18. The method of claim 15 wherein the retaining fluid isin a region between the packed column and a valve, and wherein theregion is void of matrix material as the packed column is formed.
 19. Amethod of purifying a component of a sample, comprising: providing acolumn chamber, the column chamber having an inlet end and an outletend, the outlet end terminating proximate both a first flow path and asecond flow path, the first flow path being obstructed with a porousretaining material permeable to a first fluid and impermeable to amatrix material, and the second flow path being blocked by a blockingmaterial that removably blocks flow of both the first fluid and thematrix material; flowing the first fluid and the matrix material intothe column chamber and along the first flow path to form a packed columnof the matrix material within the column chamber, the matrix materialbeing configured to selectively retain a component of the sample;flowing the sample through the packed column and along the first flowpath to separate the component from the rest of the sample; blacking thefirst flow path and removing the blocking material from the second flowpath without moving the column chamber; and after removing the blockingmaterial, flowing a second fluid through the column chamber and alongthe second flow path to remove the matrix material from the columnchamber.
 20. The method of claim 19 wherein the component comprises aradioactive atom.
 21. The method of claim 19 wherein the sample is abiological sample and the component comprises a nucleic acid.
 22. Themethod of claim 21 wherein the nucleic acid comprises at least one ofDNA or RNA.
 23. The method of claim 19 further comprising eluting thecomponent from the packed column before removing the matrix materialfrom the column chamber.
 24. The method of claim 19 further comprisingeluting the component from the matrix material after removing the matrixmaterial from the column chamber.
 25. The method of claim 19 furthercomprising recirculating at least some portions of the sample throughthe packed column prior to removing the matrix material from the columnchamber.
 26. The method of claim 19 wherein the blocking material isseparated from the packed column by a fluid-filled region.
 27. A methodof purifying a nucleic acid, comprising: providing a column chamber, thecolumn chamber having an inlet end and an outlet end, the outlet endterminating proximate both a first flow path and a second flow path, thefirst flow path being obstructed with a porous retaining materialpermeable to a first fluid and impermeable to a matrix material, and thesecond flow path being blocked by a blocking material that removablyblocks flow of both the first fluid and the column matrix material;flowing a mixture of the first fluid and the matrix material into thecolumn chamber and along the first flow path to form a packed column ofthe matrix material within the column chamber, the blocking materialdefining a portion of a periphery of the packed column, the matrixmaterial being configured to selectively retain a nucleic acid sequence;flowing a sample containing the nucleic acid sequence through the packedcolumn and along the first flow path to separate the nucleic acidsequence from other components of the sample; removing the blockingmaterial; and after removing the blocking material, flowing a secondfluid through the column chamber and along the second flow path toremove the matrix material from the column chamber.
 28. A column-basedseparations system, comprising: a column chamber having an inlet and anoutlet, said outlet in fluid communication with a first flow pathobstructed by a porous retaining material permeable to a carrier fluidand impermeable to a column matrix material, the first flow pathextending through a valve port controlling fluid flow and through theretaining material; and a second flow path in fluid communication withthe fluid outlet, said second flow path extending through a second valveport controlling flow of said matrix material.
 29. The system of claim28 wherein: said column chamber comprises in cross-sectional sideview apair of opposing sidewalls; said retaining material blocking the outlet,one of the opposing sidewalls terminating in a fluid-tight seal at theretaining material, the other of the sidewalls terminating at a locationelevationally displaced from the retaining material; and the secondvalve port extending to a valve that is laterally displaced from thecolumn chamber, the valve having a closed position and an open position,the valve in the closed position substantially preventing fluid andmatrix material flow under the other of the sidewalls, the valve in theopen position enabling fluid and matrix material to flow under the otherof the sidewalls.
 30. The system of claim 28 wherein a valve having atleast 2 positions and at least 4 ports includes said first and secondvalve ports.
 31. The system of claim 28 comprising: said column chamberdefining a portion of a periphery configured to retain a packed column,another portion of the periphery defined by a blocking material thatremovably blocks flow of both the carrier fluid and the column matrixmaterial, the blocking material spaced from the packed column by aregion configured to retain a fluid.
 32. The system of claim 31 whereina valve having at least 2 positions and at least 4 ports includes saidfirst and second valve ports, and wherein the blocking material isconnected to the column chamber through the valve, a first of the atleast two positions coupling the blocking material with the columnchamber to block flow of the column matrix material from the columnchamber, a second of the at least two positions uncoupling the blockingmaterial from the column chamber to remove the portion of the peripherydefined by the blocking material and thereby permit flow of the columnmatrix material from the column chamber.
 33. The system of claim 28wherein the column chamber comprises a periphery configured to retain acolumn matrix material, a portion of the periphery being defined by aretaining fluid.
 34. The system of claim 33 wherein the column chambercomprises a bend proximate the outlet, and wherein the portion of theperiphery defined by the retaining fluid is at the bend.
 35. The systemof claim 33 wherein the retaining fluid is in a liquid state.
 36. Thesystem of claim 33 wherein said retaining fluid is constrained by ablocking material that removably blocks flow of both a carrier fluid anda column matrix material, the blocking material being spaced from thepacked column by a fluid-filled region.
 37. The system of claim 36wherein the column chamber comprises a longitudinal section and a bend,the bend being proximate the outlet, wherein the fluid-filled region isa tube at the bend, and wherein the longitudinal section, tube and bendtogether define a “T” shape.
 38. The system of claim 37 wherein thelongitudinal section, tube and bend together define a “T” shape.
 39. Thesystem of claim 36 further comprising: a pair of flow paths into theinlet, a first of the pair of flow paths comprising first tubing and asecond of the pair of flow paths comprising second tubing, the firsttubing having a larger internal diameter opening than the second tubing.40. A method of purifying a component of a sample, comprising: providinga column chamber, the column chamber having an inlet end and an outletend, the outlet end terminating proximate both a first flow path and asecond flow path, the flow paths extending through a valve having atleast two positions that controls flow in the flow paths, the first flowpath being obstructed with a porous retaining material permeable to afirst fluid and impermeable to a matrix material, flow in the secondflow path blocked when the valve is in a first of the at least 2positions, and flow in the first flow path blocked when the valve is ina second of the at least two positions; placing the valve in the firstposition; flowing the first fluid and the matrix material into thecolumn chamber and along the first flow path to form a packed column ofthe matrix material within the column chamber, the matrix material beingconfigured to selectively retain a component of the sample; flowing thesample through the packed column and along the first flow path toseparate the component from the rest of the sample; placing the valve inthe second position; and flowing a second fluid through the columnchamber and along the second flow path to remove the matrix materialfrom the column chamber.
 41. The method of claim 40 wherein thecomponent comprises a radioactive atom.
 42. The method of claim 40wherein the component comprises a biological material.
 43. The method ofclaim 42 wherein the biological material is a nucleic acid.
 44. Themethod of claim 42 wherein the biological material is selected from thegroup consisting of viruses, cells, and combinations thereof.
 45. Themethod of claim 42 wherein the biological material is selected from thegroup consisting of proteins, peptides, and amino acids.
 46. The methodof claim 40 wherein the component comprises an organic material.
 47. Themethod of claim 40 wherein the component comprises an inorganicmaterial.
 48. The method of claim 40 further comprising detecting thecomponent.
 49. The method of claim 40 further comprising eluting thecomponent from the packed column before removing the matrix materialfrom the column chamber.
 50. The method of claim 40 further comprisingeluting the component from the matrix material after removing the matrixmaterial from the column chamber.
 51. The method of claim 40 furthercomprising recirculating at least some portions of the sample throughthe packed column prior to removing the matrix material from the columnchamber.
 52. The method of claim 19 wherein the component comprises abiological material.
 53. The method of claim 52 wherein the biologicalmaterial is a nucleic acid.
 54. The method of claim 52 wherein thebiological material is selected from the group consisting of viruses,cells, and combinations thereof.
 55. The method of claim 52 wherein thebiological material is selected from the group consisting of proteins,peptides, and amino acids.
 56. The method of claim 19 wherein thecomponent comprises an organic material.
 57. The method of claim 19wherein the component comprises an inorganic material.
 58. The method ofclaim 19 further comprising detecting the component.